The intermingling and bi-directional transfer of maternal and fetal cells during gestation creates a fascinating and highly relevant physiological model of immune tolerance. Although this interaction has been primarily addressed from the perspective of maternal tolerance to paternal antigens expressed by the fetus, maturing fetal cells also encounter an equally vast array of noninherited maternal antigens that are immunologically foreign. Thus, regulatory pathways are likely in place beginning at the earliest stages of immune cell development to maintain tolerance to noninherited maternal antigen (NIMA). Furthermore, these tolerance pathways are also likely to be maintained long term given the increasingly appreciated sustained microchimerism of maternal cells in offspring. In this regard, there is already compelling evidence illustrating immune recognition and tolerance to NIMA and other foreign antigens with exposure beginning in utero. However, what remains unknown are the immune cell and molecular pathways whereby tolerance to NIMA and other in utero exposed antigens is achieved. We propose key scientific bottlenecks include the inability to precisely identify NIMA responsive immune cells essential for characterizing their molecular phenotype, and the tools for discriminating these potentially rare cells in an antigen specific fashion from those with irrelevant specificity. We show in initial proof of concept feasibility studies these limitations are overcome using transgenic female mice engineered to constitutively and ubiquitously express defined model antigens for mating with non transgenic males that transforms model antigens into surrogate NIMA. In turn, newly developed tetramer staining and enrichment techniques allow endogenous immune cells with surrogate NIMA specificity to be tracked in an antigen specific fashion. Using this approach, our initial findings show CD4 T cells with NIMA specificity are (i) found in the peripheral lymphoid tissue in adult mice, (ii) selectively enriched for Foxp3 expression with >2-fold higher levels compared with naive or noninherited paternal antigen exposed controls, and (iii) expand only modestly while retaining exaggerated Foxp3 expression after infection with recombinant Listeria monocytogenes engineered to express the same NIMA associated peptide as a pathogen associated antigen. Based on these initial findings, our overall hypothesis is physiological exposure to noninherited maternal antigen in utero triggers tolerance through the selective accumulation of suppressive Foxp3+ regulatory T cells with specificity to these immunologically foreign antigens. To investigate this hypothesis, two inter-related aims are proposed to establish the precise immune cell subset that dictates NIMA induced tolerance, and define the necessity for maternal cell microchimerism in tolerance phenotypes for CD4 T cells with NIMA specificity. The successful completion of the aims for this exploratory grant will unlock and establish the scientific framework for additional more in depth functional and mechanistic studies aimed at further unraveling this under appreciated aspect of immunological tolerance.